The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and the aqueous compartment termed the periplasm that is located between the membranes. The long term goal of this grant has always been to understand the mechanisms of envelope biogenesis in molecular terms using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins (LP), and -barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified most, if not al, of the essential proteins required to transport LPS and OMPs across the periplasm and assemble these molecules in the OM. Work in the previous funding period has provided functional insights into the OM components of these two assembly machines, LptDE and BamABCDE respectively, and their associated periplasmic chaperones. Models derived from this work provide experimentally testable predictions that will guide our efforts. To address the important question of PL transport to the OM, we will develop a technique, using fluorescence microscopy, to follow PL movement between membranes in living cells. This will facilitate the investigation of conditions and mutations that affect this poorly understood, essential process. We have also identified OM lipoproteins in E. coli that are surface exposed, and we will develop methods to probe the mechanism that catalyzes this process. Our interest in envelope stress responses has led to the discovery of a novel role for PldA in the biogenesis of outer membrane vesicles and we will characterize this function. We have also discovered that the Cpx response can reduce the reactive oxygen species produced by certain antibiotics, and we will test the hypothesis that it does so by increasing the synthesis and/or the assembly of cytochrome bo3. LacZ can fold in the periplasm if DsbA is removed, and we will identify the Cpx regulon member(s) that catalyzes this folding reaction, and elucidate its normal function in periplasmic physiology. These studies will provide important information regarding the structure, function, and the interactions within, and between, the cellular components that catalyze envelope biogenesis. Multi-drug resistant Gram- negative bacteria are a growing concern. Insights obtained from this work should facilitate the design of novel antibacterial agents.